1. Field of the Invention
This invention relates to recombinant integration vectors. More specifically, this invention provides recombinant integration vectors containing sequences of a gene encoding an aldose reductase (AR), but not the entire AR gene. The recombinant vector can be used to specifically delete or disrupt the AR-encoding gene of a host cell. The recombinant vector also permits any heterologous sequence to be integrated into the host genomic AR sequence. Integration into the AR sequence of, for example a yeast strain, renders the recombinant strain less efficient at producing, or even unable to produce, xylitol from xylose. The recombinant vector can further be used to insert genes coding for xylose utilizing enzymes, which provides a recombinant strain which not only can utilize xylose but is simultaneously prevented from xylitol formation through the action of the AR.
2. Description of the Related Art
Lignocellulose is the main component of forest product residues and agricultural waste. Lignocellulosic raw materials are mainly composed of cellulose, hemicellulose, and lignin. The cellulose fraction is made up of glucose polymers, whereas the hemicellulose fraction is made up of a mixture of glucose, galactose, mannose, xylose, and arabinose polymers. The lignin fraction is a polymer of phenolic compounds.
The cellulose and hemicellulose fractions can be hydrolyzed to monomeric sugars, which can be fermented to ethanol. Ethanol can serve as an environmentally friendly liquid fuel for transportation, since carbon dioxide released in the fermentation and combustion processes will be taken up by growing plants in forests and fields.
The price for lignocellulose-derived ethanol has been estimated by von Sivers et al. (xe2x80x9cCost analysis of ethanol production from willow using recombinant Escherichia colixe2x80x9d, Biotechnol. Prog. 10:555-560, 1994). The calculations are based on the fermentation of all hexose sugars (glucose, galactose, and mannose) to ethanol. It was estimated that the fermentation of pentose sugars (xylose and arabinose) to ethanol will reduce the price of ethanol by approximately 25%. Xylose is found in hardwood hemicellulose, whereas arabinose is a component in hemicellulose in certain agricultural crops, such as corn. In order to make the price more competitive, the price must be reduced.
The release of monomeric sugars from lignocellulosic raw materials also releases by-products, such as weak acids, furans, and phenolic compounds, which are inhibitory to the fermentation process. Numerous studies have shown that the commonly used Baker""s yeast, Saccharomyces cerevisiae, is the only ethanol producing microorganism that is capable of efficiently fermenting non-detoxified lignocellulose hydrolysates (Olsson and Hahn-Hxc3xa4gerdal, xe2x80x9cFermentation of lignocellulosic hydrolysates for ethanol productionxe2x80x9d, Enzyme Microbial Technol. 18:312-331, 1996). Particularly efficient fermenting strains of S. cerevisiae have been isolated from the fermentation plant at a pulp and paper mill (Linden et al., xe2x80x9cIsolation and characterization of acetic acid-tolerant galactose-fermenting strains of Saccharomyces cerevisiae from a spent sulfite liquor fermentation plantxe2x80x9d, Appl. Environ.Microbiol.58:1661-1669, 1992).
S. cerevisiae ferments the hexose sugars glucose, galactose and mannose, but is unable to ferment the pentose sugars xylose and arabinose due to the lack of one or more enzymatic steps. S. cerevisiae can ferment xylulose, an isomerization product of xylose, to ethanol (Wang et al., xe2x80x9cFermentation of a pentose by yeastsxe2x80x9d, Biochem. Biophys. Res. Commun. 94:248-254,1980; Chiang et al., xe2x80x9cD-Xylulose fermentation to ethanol by Saccharomyces cerevisiaexe2x80x9d, Appl. Environ. Microbiol. 42:284-289,1981; Senac and Hahn-Hxc3xa4gerdal, xe2x80x9cIntermediary metabolite concentrations in xylulose- and glucose-fermenting Saccharomyces cerevisiae cellsxe2x80x9d, Appl. Environ. Microbiol. 56:120-126, 1990).
In eukaryotic cells, the initial metabolism of xylose is catalyzed by a xylose reductase (XR), which reduces xylose to xylitol, and a xylitol dehydrogenase (XDH), which oxidizes xylitol to xylulose. Xylulose is phosphorylated to xylulose 5-phosphate by a xylulose kinase (XK) and further metabolized through the pentose phosphate pathway and glycolysis to ethanol.
S. cerevisiae has been genetically engineered to metabolize and ferment xylose. The genes for XR and XDH from the xylose fermenting yeast Pichia stipitis have been expressed in S. cerevisiae (European Patent to C. Hollenberg, 1991; Hallborn et al., xe2x80x9cRecombinant yeasts containing the DNA sequences coding for xylose reductase and xylitol dehydrogenase enzymesxe2x80x9d, WO91/15588; Kxc3x6tter and Ciriacy, xe2x80x9cXylose fermentation by Saccharomyces cerevisiaexe2x80x9d, Appl. Microbiol. Biotechnol. 38:776-783, 1993). The transformants metabolize xylose but do not ferment the pentose sugar to ;ethanol.
When the gene for the enzyme transaldolase (TAL) is overexpressed in xylose- metabolizing transformants, the new recombinant strains grow better on xylose but still do not produce any ethanol from xylose (Walfridsson et al., xe2x80x9cXylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TAL1genes encoding the pentose phosphate pathway enzymes transketolase and transaldolasexe2x80x9d, Appl. Environ. Microbiol. 61:4184-4190, 1995). In these strains, the major metabolic by-product, in addition to cell mass, is xylitol formed from xylose through the action of the enzyme XR. When the expression of XDH is ten times higher than the expression of XR, xylitol formation is reduced to zero (Walfridsson et al., xe2x80x9cExpression of different levels of enzymes from Pichia stipitis XYL1 and XYL2 genes in s and its effect on product formation during xylose utilizationxe2x80x9d, Appl. Microbiol. Biotechnol. 48:218-224, 1997). However, xylose is still not fermented to ethanol.
The gene for xylulose kinase (XK) from S. cerevisiae has been cloned and overexpressed in XR-XDH-expressing transformants of S. cerevisiae (Deng and Ho, xe2x80x9cXylulokinase activity in various yeasts including Saccharomyces cerevisiae containing the cloned xylulokinase genexe2x80x9d, Appl. Biochem. Biotechnol. 24/25:193-199, 1990; Ho and Tsao, xe2x80x9cRecombinant yeasts for effective fermentation of glucose and xylosexe2x80x9d, WO95/13362, 1995; Moniruzzaman et al., xe2x80x9cFermentation of corn fibre sugars by an engineered xylose utilizing Saccharomyces strainxe2x80x9d, World J. Microbiol. Biotechnol. 13:341-346,1997). These strains have been shown to produce net quantities of ethanol in fermentations of mixtures of xylose and glucose. Using the well established ribosomal integration protocol, the genes have been chromosomally integrated to generate strains that can be used in complex media without selection pressure (Ho and Chen, xe2x80x9cStable recombinant yeasts for fermenting xylose to ethanolxe2x80x9d, WO97/42307; Toon et al., xe2x80x9cEnhanced cofermentation of glucose and xylose by recombinant Saccharomyces yeast strains in batch and continuous operating modesxe2x80x9d, Appl. Biochem. Biotechnol. 63/65:243-255, 1997).
In prokaryotic cells, xylose is isomerized to xylulose by a xylose isomerase (Xl). Xylulose is further metabolized in the same manner as in the eukaryotic cells. Xl from the thermophilic bacterium Thermus thermophilus was expressed in S. cerevisiae, and the recombinant strain fermented xylose to ethanol (Walfridsson et al., xe2x80x9cEthanolic fermentation of xylose with Saccharomyces cerevisiae harboring the Thermus thermophilus xylA gene which expresses an active xylose (glucose) isomerasexe2x80x9d, Appl. Environ. Microbiol. 62:4648-4651, 1996). The low level of ethanol produced was assumed to be due to the fact that the temperature optimum of the enzyme is 85xc2x0 C., whereas the optimum temperature for a yeast fermentation is 30xc2x0 C.
Recently, the gene for Xl from a mesophilic bacterium, Streptomyces diastaticus, has been cloned and transformed into S. cerevisiae. When xylose is fermented by an Xl expressing transformant of S. cerevisiae, a considerable amount of xylitol is formed in addition to ethanol. The xylitol is believed to be produced by an unspecified aldose reductase (AR) (Kuhn et al., xe2x80x9cPurification of an aldo-keto reductase from Saccharomyces cerevisiaexe2x80x9d, Appl. Environ. Microbiol. 61:1580-1585, 1995).
Although great strides have been made, there exists a need in the art for a method of efficiently fermenting lignocellulose hydrolysates to produce ethanol.
In order fulfill the above-noted need, the present invention provides genetically engineered (recombinant) expression vectors, and recombinant cells capable of fermenting lignocellulose hydrolysates to ethanol. This invention aids in fulfilling a need in the art by providing integration vectors containing sequences of an aldose reductase (AR). The recombinant vectors can be used to specifically delete or disrupt an endogenous AR gene, and can also be used to incorporate heterologous polynucleotide sequences into host (recipient) cells. The recombinant vector constructs permit any gene, including those coding for xylose-utilizing enzymes, to be integrated in the AR gene sequence. In this way, recombinant cells are produced that show enhanced conversion of xylose to ethanol while simultaneously showing reduced xylitol formation through the action of the endogenous AR.
Accordingly, the invention provides methods of making recombinant cells and methods of efficiently producing ethanol from lignocellulose-containing compositions.